Typical fluorescence based optical analysis of analytical reactions employs reactants or other reagents in the reaction of interest that bear a fluorescent moiety, such as a labeling group, where the detection of that moiety is indicative of a particular reaction result or condition. For example, reactions may be engineered to produce a change in the amount, location, spectrum, or other characteristic upon occurrence of a reaction of interest.
During analysis, an excitation light source is directed through an optical system or train at the reaction to excite fluorescence from the fluorescent moiety. The emitted fluorescence is then collected by the optical train and directed toward a detection system, which quantifies, records, and/or processes the signal data from the fluorescence. Fluorescence-based systems are generally desired for their high signal levels deriving from the high quantum efficiency of the available fluorescent dye moieties. Because of these high signal levels, relatively low levels of the materials are generally required in order to observe a fluorescent signal.
Notwithstanding the great benefits of fluorescent reaction systems, the application of these systems does have some drawbacks particularly when used in extremely low signal level reactions, e.g., low concentration or even single molecule detection systems. In particular, these systems often have a number of components that can potentially generate amounts of background signal, e.g., detected signal that does not emanate from the fluorescent species of interest, when illuminated with relatively high intensity radiation. This background signal can contribute to signal noise levels, and potentially overwhelm relatively low reaction derived signals or make more difficult the identification of signal events, e.g., increases, decreases, pulses etc., of fluorescent signal associated with the reactions being observed.
Background signal, or noise, can derive from a number of sources, including, for example, fluorescent signals from non targeted reaction regions, fluorescence from targeted reaction regions but that derive from non-relevant sources, such as non-specific reactions or associations, such as dye or label molecules that have nonspecifically adsorbed to surfaces, prevalence or build up of labeled reaction products, other fluorescent reaction components, contaminants, and the like. Other sources of background signals in fluorescent systems include signal noise that derives from the use of relatively high-intensity excitation radiation in conjunction with sensitive light detection. Such noise sources include those that derive from errant light entering the detection system that may come from inappropriately filtered or blocked excitation radiation, and/or contaminating ambient light sources that may impact the overall system. Other sources of signal noise resulting from the application of high intensity excitation illumination derives from the auto-fluorescence of the various components of the system when subjected to such illumination, as well as Raman scattering of the excitation illumination. The contribution of this systemic fluorescence is generally referred to herein as autofluorescence background noise (ABN).
It would be therefore desirable to provide methods, components and systems in which background signal, such as autofluorescence background noise, was minimized. This is particularly the case in relatively low signal level reactions, such as single molecule fluorescence detection methods and systems. The present invention meets these and other needs.